Cloning, Expression, and Purification of Recombinant Proteins
Expression and purification of yeast E1 (Aos1/Uba2), E2 (Ubc9), yeast SUMO (NΔ18 Smt3), Siz1112–465
and affinity tag-free yeast PCNA (Wt, K127G and FLKI125–128
AAAA) have been described22,23
. PCNA K164R and SUMO K19R were generated by PCR-based site-directed mutagenesis. Srs21027–1174
was obtained by PCR from S. cerevisiae
genomic DNA and cloned into the pSMT3 vector encoding an N-terminal His6
-Smt3 tag. Fragments, including the Srs21107–1174
construct, and mutants were generated by subcloning and by PCR-based site-directed mutagenesis. Srs2 was expressed in BL21 (DE3) RIL Codon Plus cells after induction with 0.5 mM IPTG and incubation of the cultures at 30°C for 3 hours. Cell pellets were suspended in 2.5× volume of 20% sucrose, 50 mM Tris, pH 8.0 and lysed by sonication. Srs2 was isolated from cleared lysate by immobilized metal affinity chromatography using Ni-NTA resin (Qiagen). Srs2 was eluted from the resin in buffer containing 250 mM imidazole, 350 mM NaCl, 20 mM Tris, pH 8.0, 1 mM βME, and the His6
-Smt3 tag was subsequently cleaved by addition of Ulp1 to a 1:1000 mass ratio followed by overnight incubation/dialysis into 350 mM NaCl, 20 mM Tris, pH 8.0, 1 mM βME at 4°C40
. Ulp1 was inactivated by adding NEM to a final concentration of 5 mM followed by incubation at RT for 30 min and 4°C for 1 hour. Srs2 was separated from other proteins in this mixture by size-exclusion chromatography (Superdex75 26/60; GE Healthcare; equilibrated with 350 mM NaCl, 20 mM Tris, pH 8.0, 1 mM βME). Fractions containing Srs2 were combined, dialyzed overnight against 50 mM NaCl, 20 mM Tris, pH 8.0, 1 mM βME and purified by anion exchange chromatography (MonoQ 10/10; GE Healthcare). Srs21107–1174
was recovered in the unbound fraction, and Srs21027–1174
was eluted using a gradient from 50 mM to 475 mM NaCl over 12 column volumes in 20 mM Tris, pH 8.0, 1 mM βME. Srs2 was concentrated using an Amicon Ultra centrifugal filter, snap frozen in liquid nitrogen and stored at −80°C. Eco1 was cloned from S. cerevisiae
genomic DNA into the pET28a vector encoding an N-terminal hexahistidine tag. Eco1 was expressed in BL21 (DE3) RIL Codon Plus cells and purified through IMAC steps according to the same protocols described for expression and purification of Srs2 fragments. Upon elution from the Ni-NTA resin, Eco1 was purified by size-exclusion chromatography (Superdex75 26/60; GE Healthcare; equilibrated with 350 mM NaCl, 20 mM Tris, pH 8.0, 1 mM βME). Peak fractions were combined and Eco1 was concentrated to 8 mg/mL prior to snap freezing in liquid nitrogen for storage at −80°C.
Reductive methylation of SUMO and preparation of PCNAmono
Reductive methylation of yeast SUMO (NΔ18 K19R Smt3;*
SUMO where the asterisk indicates reductively methylated) utilized paraformaldehyde and sodium borohydride as the methyl donor and reducing agent, respectively41
. Monomeric PCNA or monomeric SUMO-PCNA was prepared by adding N-ethylmaleimide (NEM) to a final concentration of 5 mM to the corresponding trimeric species, incubating for 2 hours at 4°C, and then purifying by size exclusion chromatography (Superdex200 26/60).
Reconstitution of SUMO-PCNA
Yeast SUMO was conjugated to yeast PCNA using the SUMO E3 ligase Siz1 by mixing 15 µM PCNA, 65 µM SUMO (NΔ18 K19R Smt3 or reductively methylated NΔ18 K19R Smt3), 90 nM Aos1/His6-Uba2 ΔCT (E1), 300 nM Ubc9 (E2), and 2 µM Siz1112–465 (E3) and 2 mM ATP, incubated at 37°C for 2 hours, and stopped by chromatographic resolution of the reaction components.
Fluorescence Polarization Assays
A cysteine residue was added N-terminal to Srs21107–1174
which itself lacked any cysteine residues. BODIPY FL (Invitrogen) was conjugated to the thiol group via maleimide chemistry. For FP assays, substrate was titrated against a fixed concentration (20 nM) of labeled Srs21107–1174
by mixing equal volumes of Srs2 peptide and serially diluted protein. Binding reaction conditions were 50 mM NaCl, 20 mM Tris, pH 8.0, 4 mM DTT. 20 µL reaction volumes were added to the wells of a 384-well microplate, and measurements taken using a SpectraMax M5 microplate reader (Molecular Devices) employing an excitation wavelength of 485 nm, emission wavelength of 538 nm, and a cutoff of 530 nm. Experiments were performed in triplicate. Using the software package Prism 5 (GraphPad Software, Inc.) data were fit to a single site binding model accounting for ligand depletion:
where A is the measured anisotropy, [Srs2] is the fixed concentration of labeled receptor, and [Prot] is the total concentration of substrate. Ab
are limiting anisotropies for bound and free peptide, respectively, and Kd
is the dissociation constant.
For displacement assays, BODIPY labeled Srs21107–1174 at 40 nM was pre-incubated with PCNAtri K127G, GST-SUMO, or SUMOK164-PCNAtri at 600, 2000, and 80 nM, respectively. Equal volumes of the pre-incubated binding reaction and serially diluted unlabled Srs21107–1174 were then mixed. 20 µL reaction volumes were added to the wells of a 384-well microplate, and measurements were made as for the direct FP assays. Experiments were performed in triplicate, and the data were fit using Prism 5 to a one site binding model for competition assays.
Assessment of Protein-Protein Interactions by Analytical Gel Filtration
Purified proteins were mixed at the indicated concentrations in a volume of 220 µL and centrifuged, and 200 µL was loaded onto a Superose12 HR 10/30 column (GE Healthcare) equilibrated in 50 mM NaCl, 20 mM Tris, pH 8.0, 1 mM βME. The column was eluted at a flow rate of 0.5 mL/min, and 0.5 mL fractions were collected, analyzed by SDS-PAGE and stained with Sypro Ruby Protein Gel Stain (Bio-Rad).
Assessment of Protein-Protein Interactions by Pull-downs
400 µL 750 µg/mL His6-Eco1 in binding buffer (200 mM NaCl, 20 mM Tris, pH 8.0, 40 mM imidazole) was added to 50 µL of 50% (v/v) Ni-NTA resin (Qiagen) equilibrated with the same buffer and mixed for 20 min at 4°C. Beads were subsequently washed with 3 × 400 µL of binding buffer to remove unbound His6-Eco1. 400 µL of 10 µM prey protein in binding buffer was added to Eco1 coated beads and mixed for 15 min at 4°C. Unbound prey protein was removed with 3 × 400 µL washes with binding buffer. Material was eluted from beads with successive 400 µL washes of binding buffer that contained increasing concentrations of NaCl. Eco1 and any remaining bound prey protein was eluted in a final wash with 100 µL 350 mM NaCl, 20 mM Tris, pH 8.0, 400 mM imidazole.
Crystallization and Structure Determination
Crystals were grown using the hanging drop vapor diffusion method by mixing equal volumes of sample and reservoir solution. SUMOK164-PCNAtri K127G: Purified SUMOK164-PCNAtri was crystallized at 18°C in 4% PEG 8000, 500 mM LiSO4 (pH 4.5). Crystals were transferred to cryoprotectant (reservoir supplemented with 15% ethylene glycol) prior to flash freezing in LN2. Diffraction data were collected at beamline 24-ID-C at the Advanced Photon Source at a wavelength of 0.979 Å. Crystals belong to space group F432 with one protomer of SUMO-PCNA in the asymmetric unit. SUMOK164-PCNAmono K127G: Srs21027–1174 was added to 8 mg/mL SUMOK164-PCNA in 3-fold molar excess and dialyzed against 100 mM NaCl, 20 mM Tris pH 8.0, 1 mM βME at 4°C for 16 hrs. The Srs21027–1174:SUMOK164-PCNA complex was purified on a Superdex 200 column and concentrated to ~8 mg/mL. Crystals were obtained at 4°C in 21% MPD, 100 mM BaCl2, 100 mM Bis-Tris, pH 6.5 and transferred to cryoprotectant (reservoir supplemented with 12% glycerol) prior to flash freezing in LN2. Diffraction data were collected at beamline 24-ID-C at the Advanced Photon Source at a wavelength of 0.979 Å. Crystals belong to space group I41 with one protomer of SUMOK164-PCNAmono in the asymmetric unit. It was subsequently determined that these crystals lacked the Srs21027–1174 protein component, and that contaminating NEM, presumably present in earlier Srs1027–1174 preparations, had modified PCNA C22 and C81. SUMOK164-PCNA K127G:Srs21107–1174: Srs21107–1174 was added in 3-fold molar excess to SUMOK164-PCNAtri at a protomer concentration of 60 µM. NEM was added to a final concentration of 5 mM, and the sample incubated for 3 hours at 4°C prior to co-purification of the complex by gel filtration (Superdex200 26/60; GE Healthcare; equilibrated with 100 mM NaCl, 20 mM Tris, pH 8.0, 1 mM βME). The complex was concentrated to ~10 mg/mL and crystallized at 6°C in 1.9 M ammonium sulfate, 4% PEG400, 100 mM HEPES, pH 7.5 by the sitting drop vapor diffusion method. Crystals were washed and cryoprotected in 2 M ammonium sulfate, 4% PEG400, 100 mM HEPES, pH 7.5, 20% glycerol. Diffraction data were collected at beamline X29 of the National Synchrotron Light Source at a wavelength of 1.075 Å. Crystals belong to space group C2 with two Srs2/SUMOK164-PCNA complexes in the asymmetric unit.
Crystallographic data were indexed, integrated, and scaled using HKL200042
and data reduced using programs in CCP443
. Data obtained for SUMOK164
were scaled maintaining separation of anomalous pairs to maintain the anomalous signal from the associated barium ions. The programs MOLREP44
were used to find molecular replacement solutions using the coordinates of a yeast PCNA protomer from PDB 1PLQ and yeast SUMO from PDB 1EUV (Supplemental Table 2
and Methods). Models were manually inspected and rebuilt prior to refinement using the program O46
and refined using CCP4’s REFMAC47
. Models have reasonable geometry at the respective resolution as assessed by MolProbity48
has 95.5% and 3.9% of modeled residues in favored and allowed Ramachandran regions, respectively with 0.6% outliers. SUMOK164
has 95.7% of modeled residues in favored regions, 3.4% in allowed regions and 0.9% as outliers, and Srs21107–1174
has 91.5% in favored regions, 7.6% in allowed regions and 0.9% as outliers. Structures of SUMOK164
have Clash Scores in the 93rd
percentiles, respectively, and MolProbity Scores in the 70th
percentiles respectively. All graphical representations of structure were generated using PYMOL49
Yeast complementation and growth assays
Yeast strains used in this study include srs2Δrad6Δ (MATa, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0, srs2Δ::kanMX, rad6Δ::kanMX) and srs2Δrad18Δ (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, srs2Δ::kanMX, rad18Δ::kanMX). Double deletion strains were constructed by mating srs2Δ (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) to rad6Δ or rad18Δ (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) knockout strains from the Saccharomyces Genome Deletion Project (Open Biosystems). Diploids were selected on media lacking lysine and methionine then sporulated in 2% potassium acetate. The desired double deletion strains were selected by screening spores by PCR to establish the genotype and by replica plating spore colonies on selective media. Double deletion strains were transformed with the pRS416 plasmid (URA3) or the the pRS416 plasmid (URA3) containing SRS2 or srs2 alleles flanked by 500 bp of its endogenous 5’ and 3’ UTRs. These constructs were generated by cloning the SRS2 5' UTR along with the SRS2 coding sequence corresponding to the first 1115 residues into the XhoI and SalI sites of the pRS416 vector and the SRS2 3' UTR into the XmaI and BamHI sites. Use of the SalI site introduced a DNA mutation that is silent with respect to the encoded Srs2 amino acid. The remainder of the SRS2 coding sequence was placed between the SalI and XmaI sites to produce the desired alleles including SRS2, srs2-ΔPIPΔSIM (Srs21–1148), srs2-ΔC22 (Srs21–1152), srs2-ΔC13 (Srs21–1161), and srs2-ΔSIM (Srs21–1167). srs2-ΔPIP (Srs21–1148+PG+1168–1174) was generated by placing the SRS2 coding region corresponding to the C-terminal 7 residues along with the 3' UTR into XmaI and BamHI sites. The SRS2 coding sequence corresponding to residues 1115–1148 was then placed between the SalI and XmaI sites. This strategy introduced two non-native amino acids (Pro-Gly) between Srs2 residues 1148 and 1168. Yeast transformants were selected on media lacking uracil. For spotting assays to assess growth defects and drug sensitivity, cultures were grown overnight and diluted to an A600 of 1.0. 10-fold serial dilutions were made and 3 µl aliquots were spotted onto SD -Ura plates or SD -Ura + MMS plates containing the indicated concentration of DNA damage agent. Plates were incubated at 30°C for ~3 days with photographs taken every 6–8 hours.